The organization of cardiac muscle fiber has been extensively studied but there still remain many questions of basic importance that may be best studied by immunocytochemical examinations of semi-thin and ultrathin frozen sections of the fiber. It is possible that in the minute heart of the early embryonic stage, some of the myofibrils form three dimensional networks and perform the function similar to that of the fibers in the adult heart. The branching feature of myofibrils in the adult heart could indeed be a manifestation of this early three dimensionality of myofibril arrangement. In fact, contrary to past reports, our preliminary EM studies indicate that many myofibrils form organized networks at least locally in the earliest functional heart. We would like to study 1) the possibility that well organized myofibril networks are present throughout the embryonic heart of the earliest functional stage, 2) the relationship between the networks of myofibrils and those of intermediate filaments (IF), 3) the relationship between the myofibril networks and the distribution of fasciae adherentes, 4) the mechanisms by which the myofibrils elongate under the critical constraint that the heart must always be functional, 5) the synthesis and the distribution of the collagens, and 6) the changes in the distributions of developmentally regulated proteins. In order to visualize these structures in the light microscope, we will apply our methods of immunofluorescent labeling of semithin (0.5-1 Mum) frozen sections of developing chick embryonic heart. Fluorescein and rhodamine fluorophores will be used for double immunolabeling to study the relationship between any pair of these structures. We would also like to apply our methods of immunoelectron microscopy of ultrathin frozen sections to study the organization of adult cardiac muscle cell. The subjects to be studied in the immediate future will be 1) the distribution of IF, 2) the possibility that desmin exists in nonfilamentous form, 3) the development of SR and T systems and its relationship with the distribution of IF and mitochondria, and 4) whether or not tropomyosin is present inside the Z disc. If necessary, we will use ferritin and Imposil or gold particles of different sizes as the markers for double immunolabeling.